Microparticle sorting device, and method and program for sorting microparticles

ABSTRACT

Provided are a microparticle sorting device, and a method and a program for sorting microparticles capable of stabilizing sorting performance over a prolonged period of time. 
     The microparticle sorting device includes an imaging element and a controller. The imaging element obtains an image of fluid and fluid droplets at a position where the fluid discharged from an orifice which generates a fluid stream is converted into the fluid droplets. The controller controls driving voltage of an oscillation element which gives oscillation to the orifice and/or controls a position of the imaging element based on a state of the fluid in the image and/or a state of a satellite fluid droplet. The satellite fluid droplet does not include microparticles and exists between the position, where the fluid is converted into the fluid droplets, and a fluid droplet, among fluid droplets including the microparticles, which is closest to the position where the fluid is converted into the fluid droplets.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit under 35U.S.C. § 120 of U.S. patent application Ser. No. 14/386,368, titled“MICROPARTICLE SORTING DEVICE, AND METHOD AND PROGRAM FOR SORTINGMICROPARTICLES,” filed on Sep. 19, 2014, which is the National Stage ofInternational Application No. PCT/JP2013/081152, filed in the JapanesePatent Office as a Receiving Office on Nov. 19, 2013, which claimspriority to Japanese Patent Application No. JP 2013-124209, filed in theJapanese Patent Office on Jun. 12, 2013, and Japanese Patent ApplicationNo. 2013-013801, filed Jan. 28, 2013, which are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

The present technology relates to a microparticle sorting device, and amethod and a program for sorting microparticles. More specifically, thepresent technology relates to technology which sorts and recoversmicroparticles based on results analyzed by an optical method and thelike.

BACKGROUND ART

In the related art, an optical measuring method using a flow cytometry(flow cytometer) has been employed to analyze cells, microorganisms, andbiologically-relevant microparticles such a liposome. The flow cytometeris a device which irradiates microparticles, which flow through a flowpath disposed in a flow cell or a microchip, with light. The flowcytometer also detects fluorescence or scattered light emitted from anindividual microparticle, and analyzes the fluorescence or the scatteredlight.

Examples of the flow cytometer include a device having a function ofsorting and recovering, based on the analyzed results, onlymicroparticles which have specific characteristics. Especially, a devicewhich targets a cell as an object to be sorted is called a “cellsorter”. Generally, in this cell sorter, fluid discharged from the flowpath is converted into fluid droplets by giving oscillation to the flowcell or the microchip with an oscillation element and the like (seePatent Documents 1, 2). The fluid droplets isolated from the fluid arecharged with positive (+) or negative (−) charges. Then, travelingdirections of the fluid droplets are changed by a deflection plate.After that, the fluid droplets are recovered and put into apredetermined container and the like.

Further, a method has been suggested in the related art as technologyfor stabilizing sorting performance (see Patent Document 3). In thismethod, images of fluid or fluid droplets, which are discharged from anoutlet nozzle of a flow cell, are taken. Herein, conditions such aspressure including sheath pressure, and crystal drive are adjusteddepending on deviations calculated from the images.

CITATION LIST Patent Documents Patent Document 1: Japanese PCT NationalPublication No. 2007-532874 Patent Document 2: Japanese PatentApplication Laid-Open No. 2010-190680 Patent Document 3: Japanese PCTNational Publication No. 2006-504970 SUMMARY OF INVENTION Problems to beSolved by the Invention

However, in the above-mentioned microparticle sorting device in therelated art, there is a problem that sorting performance may be unstablebecause of an influence of differential pressure due to changes intemperature, in fluid pressure, and in sheath pressure. This problem canbe improved to some extent by taking images of fluid or fluid dropletsand adjusting various conditions based on the images, as the technologyrecited in Patent Document 3. However, in such a case, processes becomecomplicated, and at the same time, errors may easily occur in eachprocess. Such errors include, for example, a sensing error and an errorduring a change in setting pressure.

Therefore, a primary object of the present disclosure is to provide amicroparticle sorting device, and a method and a program for sortingmicroparticles capable of stabilizing sorting performance over aprolonged period of time.

Solutions to Problems

A microparticle sorting device according to the present disclosureincludes: an imaging element configured to obtain an image of fluid andfluid droplets at a position where the fluid discharged from an orificewhich generates a fluid stream is converted into the fluid droplets; anda controller configured to control driving voltage of an oscillationelement which gives oscillation to the orifice and/or control a positionof the imaging element, based on a state of the fluid in the imageand/or a state of a satellite fluid droplet which does not includemicroparticles and exists between the position, where the fluid isconverted into the fluid droplets, and a fluid droplet, among fluiddroplets including the microparticles, which is closest to the positionwhere the fluid is converted into the fluid droplets.

The controller controls, for example, the driving voltage so that adistance from a position, where the fluid is converted into the fluiddroplets, to the satellite fluid droplet and/or a state of a constrictedregion of the fluid right before being converted into the fluid dropletsbecome constant.

In cases where the state of the constricted region is controlled to beconstant, the controller may control the driving voltage so that a widthof the constricted region becomes constant.

The controller can further control the driving voltage so that adistance from the position, where the fluid is converted into the fluiddroplets, to the narrowest part of the constricted region of the fluidright before being converted into the fluid droplets becomes constant.

Moreover, the controller can also control the position of the imagingelement so that the position, in the image, where the fluid is convertedinto the fluid droplets becomes constant.

In such a case, the controller may calculate a distance from an upperend of the image to the position where the fluid is converted into thefluid droplets. The controller may further control the position of theimaging element so that the distance becomes constant.

On the other hand, this microparticle sorting device may include asheath liquid storage tank, a first water depth detector, a firstpressure detector, and a first pressure controller. The sheath liquidstorage tank stores sheath liquid included in the fluid stream. Thefirst water depth detector detects a water depth of the sheath liquidstored in the sheath liquid storage tank. The first pressure detectordetects air pressure inside the sheath liquid storage tank. The firstpressure controller controls the air pressure inside the sheath liquidstorage tank so that a sum of fluid pressure calculated from the waterdepth detected by the first water depth detector and the air pressuredetected by the first pressure detector becomes constant.

The microparticle sorting device herein may further include a sampleliquid storage tank, a second water depth detector, a second pressuredetector, and a second pressure controller. The sample liquid storagetank stores sample liquid including microparticles and included in thefluid stream. The second water depth detector detects a water depth ofthe sample liquid stored in the sample liquid storage tank. The secondpressure detector detects air pressure inside the sample liquid storagetank. The second pressure controller controls the air pressure insidethe sample liquid storage tank so that a sum of the fluid pressurecalculated from the water depth detected by the second water depthdetector and the air pressure detected by the second pressure detectorbecomes constant.

In a method for sorting microparticles according to the presentdisclosure, the driving voltage of the oscillation element which givesoscillation to the orifice and/or the position of the imaging elementwhich obtains the image are controlled, based on the state of the fluidin the image taken at the position where the fluid discharged from theorifice which generates the fluid stream is converted into the fluiddroplets and/or the state of the satellite fluid droplet which does notinclude microparticles and exists between the position, where the fluidis converted into the fluid droplets, and the fluid droplet, among thefluid droplets including the microparticles, which is closest to theposition where the fluid is converted into the fluid droplets.

A program according to the present disclosure causes the controller ofthe microparticle sorting device to execute a function of controllingthe driving voltage of the oscillation element which gives oscillationto the orifice and/or the position of the imaging element which obtainsthe image, based on the state in the image taken at the position wherethe fluid discharged from the orifice which generates the fluid streamis converted into the fluid droplets and/or the state of the satellitefluid droplet which does not include microparticles and exists betweenthe position, where the fluid is converted into the fluid droplets, andthe fluid droplet, among the fluid droplets including themicroparticles, which is closest to the position where the fluid isconverted into the fluid droplets.

Effects of the Invention

According to the present disclosure, sorting performance can bestabilized over a prolonged period of time.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view showing a configuration of a microparticlesorting device according to a first embodiment of the presentdisclosure.

FIG. 2 is a schematic view showing an exemplary image taken by a camera7 shown in FIG. 1.

FIG. 3 is a view showing a relation between states of fluid and fluiddroplets, and each parameter.

FIGS. 4A and 4B are views showing a relation between driving voltage ofan oscillation element 3 and a liquid column length.

FIGS. 5A to 5C are views showing a relation between the driving voltageof the oscillation element 3, and a first satellite upper interval d anda liquid column constricted width w.

FIG. 6 is a view showing a relation between states of fluid and eachparameter.

FIGS. 7A to 7C are views showing a relation between the driving voltageof the oscillation element 3 and a final fluid droplet length m in aliquid column.

FIGS. 8A and 8B are schematic views showing an anomaly detecting methodbased on a liquid column length L.

FIGS. 9A and 9B are views showing a status change in a fluid stream dueto a change in environmental temperature.

FIGS. 10A and 10B are views showing a method for moving a position ofthe camera 7 as a position of a break-off point changes.

FIG. 11 is a view showing other methods for maintaining a constantposition of the break-off point.

FIG. 12 is a schematic view showing an overall configuration of amicroparticle sorting device according to a second embodiment of thepresent disclosure.

FIGS. 13A and 13B are views showing a relation between sheath pressure,and a state of fluid and fluid droplets.

FIG. 14 is a schematic view showing air pressure and water pressureinside a sheath container 10.

FIG. 15 is a view showing a method for measuring a water depthD_(sheath) of sheath liquid 231.

FIG. 16 is a view showing a method for measuring a water depthD_(sample) of sample liquid 221.

FIGS. 17A and 17B are views showing a method for controlling the sheathpressure.

MODES FOR CARRYING OUT THE INVENTION

Hereinafter, modes for carrying out the present disclosure will bedescribed in detail with reference to the accompanying drawings. Notethat the present disclosure is not restricted to each embodimenthereinafter described. Further, the embodiments will be described in thefollowing order.

1. First Embodiment

(An example of a sorting device which controls an oscillation elementand an imaging element based on a state of fluid and fluid droplets)

2. Second Embodiment

(An example of a sorting device having a function of stabilizingpressure)

1. First Embodiment

First, a microparticle sorting device according to a first embodiment ofthe present disclosure will be described. FIG. 1 is a view showing aschematic configuration of the microparticle sorting device according tothe first embodiment of the present disclosure.

[Overall Configuration of Device]

A microparticle sorting device 1 of the present embodiment sorts andrecovers microparticles based on results analyzed by an optical method.The microparticle sorting device 1 herein includes a microchip 2, anoscillation element 3, an electrode 4 for charging, deflection plates 5a and 5 b, recovery containers 6 a to 6 c and the like, as shown inFIG. 1. Further, the microparticle sorting device 1 includes an imagingelement (camera 7) and a controller 8. The camera 7 obtains an image offluid and fluid droplets. The controller 8 controls driving voltage ofthe oscillation element 3 and/or a position of the camera 7 based on theimage taken by the camera 7.

[Microparticle]

The microparticles analyzed and sorted by the microparticle sortingdevice 1 of the present embodiment broadly include cells,microorganisms, and biologically-relevant microparticles such as aribosome, or include synthetic particles such as latex particles, gelparticles and industrial particles.

Examples of the biologically-relevant microparticles include achromosome, a ribosome, a mitochondrion, and an organelle, which areincluded in various cells. Further, examples of the cells include plantcells, animal cells, and blood cells. Moreover, examples of themicroorganisms include bacteria such as an E. coli, viruses such as atobacco mosaic virus, and fungi such as a yeast cell. Thesebiologically-relevant microparticles may include biologically-relevantpolymers such as nucleic acid, protein and a complex thereof.

On the other hand, examples of the industrial particles includeparticles including organic polymer materials, inorganic materials ormetallic materials. As the organic polymer materials, for example,polystyrene, styrene divinyl benzene, and polymethyl methacrylate can beused. As the inorganic materials, for example, glass, silica, andmagnetic materials can be used. As the metallic materials, for example,gold colloid and aluminum can be used. Note that these microparticlesgenerally have a spherical shape, but may have a non-spherical shape.Further, the size and the mass thereof are not specifically restricted.

[Microchip 2]

The microchip 2 includes a sample inlet 22, a sheath inlet 23, a suctionoutlet 24 and the like. Liquid (sample liquid) including microparticlesto be sorted is introduced to the sample inlet 22, while sheath liquidis introduced to the sheath inlet 23. The suction outlet 24 is foreliminating obstructions and bubbles. In this microchip 2, the sampleliquid is introduced to the sample inlet 22 and joins the sheath liquidintroduced to the sheath inlet 23. Then, the sample liquid is sent to asample flow path and is discharged from an orifice 21 disposed at an endof the sample flow path.

To the sample flow path, a suction flow path which communicates with thesuction outlet 24 is connected. When obstructions and bubbles appear inthe sample flow path, this suction flow path applies negative pressureupon the inner side of the sample flow path to reverse the flow in thesample flow path temporarily in order to eliminate the obstructions andbubbles. A negative pressure source such as a vacuum pump is connectedto the suction outlet 24.

The microchip 2 can be formed of glass or various plastics (such as PP,PC, COP, and PDMS). A preferable material for the microchip 2 is onewhich transmits measurement light emitted from a light detector, and haslittle autofluorescence as well as few optical errors due to smallwavelength dispersion.

The microchip 2 can be formed by wet etching or dry etching of a glasssubstrate, or by nanoimprinting, mold injection, or a mechanical processof a plastic substrate. The microchip 2 can be formed, for example, bysealing a substrate, on which a sample flow path and the like areformed, with a substrate including a similar or a different material.

[Oscillation Element 3]

The oscillation element 3 is abutted on a part of the microchip 2 ordisposed as an inner constituent of the microchip 2. The oscillationelement 3 gives minute oscillation to the sheath liquid by oscillatingthe microchip 2 at a predetermined frequency. Then, the oscillationelement 3 converts the fluid (sample liquid and sheath liquid)discharged from the orifice 21 into fluid droplets to generate a fluidstream (a fluid droplet stream) S. As the oscillation element 3, a piezoelement and the like can be used.

[Voltage Supplier 31]

A voltage supplier 31 supplies driving voltage to the oscillationelement 3. The driving voltage of the oscillation element 3 is suppliedin accordance with a sine wave in order to form stable fluid droplets,and is controlled by both a frequency (clock value) and amplitude (drivevalue).

[Charged Section]

A charged section applies positive or negative charges to fluid dropletsdischarged from the orifice 21. The charged section herein includes, forexample, the electrode 4 for charging and a voltage source which appliespredetermined voltage to this electrode 4 for charging. The electrode 4for charging is disposed while being brought into contact with thesheath liquid and/or the sample liquid, which flow through the flowpath. The electrode 4 for charging herein further charges the sheathliquid and/or the sample liquid, and is inserted, for example, into acharged electrode inlet of the microchip 2.

In FIG. 1, the electrode 4 for charging is disposed so as to come intocontact with the sample liquid. However, the present disclosure is notrestricted thereto and the electrode 4 for charging may be disposed soas to come into contact with the sheath liquid or with both the sampleliquid and the sheath liquid. However, it should be noted that theelectrode 4 for charging is preferably disposed so as to come intocontact with the sheath liquid, considering an influence on a cell to besorted.

In this manner, by applying the positive or negative charges to desiredfluid droplets and charging the fluid droplets, an arbitrary fluiddroplet including microparticles can be isolated by electrical force.Further, when charging timing by the charged section is synchronizedwith supply of voltage to the oscillation element 3, only the arbitraryfluid droplet can be charged.

[Deflection Plates 5 a, 5 b]

The deflection plates 5 a, 5 b change a traveling direction of eachfluid droplet within the fluid stream S by the electric force that actsbetween the deflection plates and the charges applied to the fluiddroplets. The deflection plates 5 a, 5 b herein guide the travelingdirection of each fluid droplet to a predetermined recovery containerand are disposed while facing each other across the fluid stream S. Asthese deflection plates 5 a, 5 b, for example, electrodes which aregenerally used can be employed.

The positive voltage and the negative voltage are respectively appliedto the deflection plates 5 a and 5 b. When charged fluid droplets passthrough an electric field formed by the above-mentioned voltageapplication, electric force (coulomb force) occurs, and each fluiddroplet is drawn in a direction of the deflection plate 5 a or 5 b. Themicroparticle sorting device 1 can control a direction of the fluiddroplet stream (side stream) drawn to the electric field, by changingthe polarity (positive or negative) or amount of charges applied to thefluid droplets. Therefore, a plurality of mutually differentmicroparticles can be simultaneously sorted.

[Recovery Containers 6 a to 6 c]

The recovery containers 6 a to 6 c recover fluid droplets which havepassed between the deflection plates 5 a and 5 b. As the recoverycontainers 6 a to 6 c for experiment, general-purpose plastic tubes orglass tubes can be used. It is preferable to switchably dispose theserecovery containers 6 a to 6 c inside the device. Further, among theserecovery containers 6 a to 6 c, one that receives microparticles whichare not to be sorted may be coupled to a drainage path for the recoveredfluid droplets.

Note that the number of the recovery containers disposed in themicroparticle sorting device 1 is not specifically restricted. Forexample, in cases where more than three recovery containers aredisposed, each fluid droplet may be guided to any one of those recoverycontainers and may be recovered depending on the presence or absence ofelectric acting force between the deflection plates 5 a and 5 b, and themagnitude of the force.

[Imaging Element (Camera) 7]

The imaging element (camera) 7 takes an image of the fluid before beingconverted into the fluid droplets and of the fluid droplets at aposition (break-off point BP) where a laminar flow of the sample liquidand the sheath liquid discharged from the orifice 21 is converted intofluid droplets. Note that the image of the fluid and the fluid dropletscan be taken by using various imaging elements such as a photoelectricconversion element other than the imaging device such as a CCD cameraand a CMOS camera.

Further, the camera 7 preferably includes a position adjustmentmechanism 70 in order to change the position of the camera 7. As aresult, the position of the camera 7 can be easily controlled based oncommands from the controller 8 (hereinafter described). Themicroparticle sorting device 1 according to the present embodiment mayinclude not only the camera 7 but also a light source (not shown) whichilluminates an imaging region.

[Controller 8]

The controller 8 controls driving electricity of the oscillation element3 and/or the position of the camera 7 based on the image taken by thecamera 7. More specifically, the controller 8 controls the voltagesupplier 31 and the position adjustment mechanism 70 based on the stateof the fluid, in the image, before being converted into the fluiddroplets and/or based on a state of a satellite fluid droplet existingbetween the break-off point and the fluid droplet includingmicroparticles.

The controller 8 may include, for example, an information processingdevice including a general-purpose processor, a main storage, asecondary storage and the like. In such a case, the voltage supplier 31and the position adjustment mechanism 70 can be automatically controlledby inputting, to the controller 8, image data taken by the imagingelement such as the camera 7, and by executing control algorithm whichhas been programmed. Such a computer program may be stored, for example,in a recording medium such as a magnetic disc, an optical disc, amagneto-optical disc, and a flash memory, or may be delivered through anetwork.

[Light Detector]

Further, the microparticle sorting device 1 according to the presentembodiment includes, for example, the light detector (not shown) whichirradiates a predetermined part of the sample flow path with light(measurement light) and detects light (light to be measured) emittedfrom microparticles which pass through the sample flow path. The lightdetector herein can be configured similarly to a flow cytometry in therelated art. More specifically, the light detector herein includes alaser light source, an irradiating system, and a detecting system. Theirradiating system includes, for example, a condenser lens whichcondenses a laser beam and irradiates microparticles with the laserbeam, a dichroic mirror, and a band-pass filter. The detecting systemdetects the light to be measured which is emitted from themicroparticles due to the laser beam irradiation.

The detecting system includes, for example, a photo multiplier tube(PMT) and an area imaging element such as a CCD, and a CMOS element.Note that the irradiating system and the detecting system may includethe same single optical path or may separately include an individualoptical path. Further, the light to be measured which is detected by thedetecting system of the light detector is light emitted from themicroparticles due to irradiation of the measurement light. For example,the light to be measured may be scattered light such asforward-scattered light, side-scattered light, Rayleigh scattered lightand Mie scattered light, or fluorescence. The light to be measured isconverted into electric signals. Optical characteristics of themicroparticles are detected based on the electric signals.

[Movement]

Next, a movement of the microparticle sorting device 1 according to thepresent embodiment will be described. When microparticles are sorted bythe microparticle sorting device 1 according to the present embodiment,the sample liquid including the microparticles to be sorted isintroduced to the sample inlet 22, while the sheath liquid is introducedto the sheath inlet 23. Further, the optical characteristics of themicroparticles as well as velocity (flow speed) of the microparticlesand intervals of the microparticles are detected at the same time, forexample, by using the light detector. The detected opticalcharacteristics, flow speed, intervals of the microparticles and thelike are converted into electric signals and are output to the wholecontroller (not shown) of the device.

The laminar flow of the sample liquid and the sheath liquid passesthrough a part to be irradiated with light in the sample flow path.Then, the laminar flow is discharged from the orifice 21 to a spaceoutside the microchip 2. On this occasion, the orifice 21 is oscillatedby the oscillation element 3, and fluid to be discharged is convertedinto fluid droplets. The traveling direction of each fluid dropletcharged in the sample flow path is changed by the deflection plates 5 a,5 b based on the detected results from the light detector. Then, eachfluid droplet is guided to the predetermined recovery containers 6 a to6 c and recovered.

In the series of processes, the microparticle sorting device 1 accordingto the present embodiment obtains the image of the fluid and the fluiddroplets at the break-off point by using the camera 7. Then, themicroparticle sorting device 1 controls the oscillation element 3 andthe camera 7 by using the controller 8 based on the image. Morespecifically, the controller 8 controls the driving voltage suppliedfrom the voltage supplier 31 and/or the position of the camera 7 basedon the state of the fluid in the image and/or the state of the satellitefluid droplet.

(Imaging Process)

FIG. 2 is a schematic view showing an exemplary image taken by thecamera 7. As shown in FIG. 2, an image 71 obtained by the camera 7includes at least a break-off point BP and a first satellite SD₁.Herein, the “break-off point BP” is a position where the fluiddischarged from the orifice 21 is converted into fluid droplets.Further, the “first satellite SD₁” herein is a satellite fluid dropletSD which does not include microparticles and exists between thebreak-off point BP and a fluid droplet D₁, among fluid droplets Dincluding the microparticles, which is closest to the break-off pointBP.

(Controlling Driving Voltage)

In cases where the driving voltage of the oscillation element 3 iscontrolled by the controller 8, for example, an image (reference image)in which the fluid and the fluid droplets are adjusted to be in the mostpreferable state is prepared in advance. Then, the driving voltage isadjusted so that an image during sorting matches with the referenceimage. The reference image and the image during sorting can be compared,for example, based on a distance d from the break-off point BP to thefirst satellite SD₁ (first satellite upper interval d), and based on awidth w of the constricted region (liquid column constricted width) ofthe fluid right before being converted into fluid droplets. FIG. 3 is aview showing a relation between the states of the fluid stream S andeach parameter.

When the first satellite upper interval d is narrower than when a fluiddroplet is stable, such a state represents that the break-off point BPand the first satellite SD₁ are getting closer. In cases where a valueof the first satellite upper interval d becomes smaller, or zero, such astate represents that a position of the break-off point BP has droppedby the first satellite SD₁ (fluid droplet unstable state shown in FIG.3).

In cases where the liquid column constricted width w is narrow, such astate represents that the liquid column is about to be cut off. In caseswhere a value of the liquid column constricted width w becomes smaller,or zero, the liquid column is completely cut off and a new fluid dropletD is formed. Such a state represents that the break-off point BP hasrisen by the newly formed fluid droplet D (fluid droplet unstable stateshown in FIG. 3).

The first satellite upper interval d, the liquid column constrictedwidth w, and a liquid column length L (the position of the break-offpoint BP) have a mutually close relation. The liquid column length L,the first satellite upper interval d, and the liquid column constrictedwidth w are indexes which directly show stability of the break-off pointBP. Based on the value of the first satellite upper interval d or thevalue of the liquid column constricted width w, a fluid droplet shape ofthe fluid stream S can be stabilized by controlling the driving voltageof the oscillation element 3.

FIGS. 4A and 4B are views showing a relation between the driving voltageof the oscillation element 3 and the liquid column length L. FIGS. 5A to5C are views showing a relation between the driving voltage of theoscillation element 3, and the first satellite upper interval d and theliquid column constricted width w. For example, in cases where the image71 shown in FIG. 2 is regarded as a reference image, the driving voltageof the oscillation element 3 is controlled by the controller 8 so thatthe liquid column length L in an image 72 during sorting becomesL_(ref)±1 (1 represents an arbitrary number of a pixel). As a result,the number of fluid droplets FD, inside the liquid column, which areincluded in the fluid right before being converted into fluid dropletsbecomes constant. The “fluid droplet inside the liquid column” hereinrepresents a fluid droplet, before being separated, which is included inthe fluid before being converted into fluid droplets.

As shown in FIGS. 4A and 4B, in cases where the driving voltage of theoscillation element 3 increases, the liquid column is cut off, and thefluid droplet FD, inside the liquid column, which is closest to thebreak-off point BP is converted into fluid droplets. As a result, theposition of the break-off point BP rises, and the value of the liquidcolumn length L decreases. By contrast, in cases where the drivingvoltage of the oscillation element 3 decreases, the first satellite SD₁becomes large and is converted into a liquid column, and into a fluiddroplet FD inside the liquid column. As a result, the position of thebreak-off point BP drops, and the value of the liquid column length Lincreases.

The controller 8 utilizes this relation to control the driving voltageof the oscillation element 3. Note that, under a state where sheath flowspeed is constant, no change occurs in fluid droplet intervals. Further,a change in the position of the break-off point BP, which is caused bythe change in the fluid droplet intervals, does not occur, either.Therefore, the driving voltage of the oscillation element 3 can becontrolled so as to meet desired conditions easily.

Next, the driving voltage of the oscillation element 3 is controlled sothat the first satellite upper interval d in the image 72 during sortingbecomes similar to a first satellite upper interval d_(ref) in thereference image 71 shown in FIG. 2. As shown in FIGS. 5A to 5C, in caseswhere the driving voltage of the oscillation element 3 increases, thevalue of the first satellite upper interval d increases. By contrast, incases where the driving voltage of the oscillation element 3 decreases,the value of the first satellite upper interval d decreases. Thecontroller 8 utilizes this relation to control the driving voltage ofthe oscillation element 3.

The first satellite upper interval d is sensitive to a change in thefluid droplet shape of the fluid stream S. Therefore, the fluid dropletshape during sorting can be maintained as stable as a state similar tothe reference image by keeping adjusting the first satellite upperinterval d so as to match with the first satellite upper intervald_(ref) of the reference image 71.

Further, instead of the above-mentioned first satellite upper intervald_(ref), the driving voltage of the oscillation element 3 can becontrolled by using the liquid column constricted width w. Morespecifically, the driving voltage of the oscillation element 3 iscontrolled so that the value of the liquid column constricted width w inthe image 72 during sorting becomes similar to a liquid columnconstricted width w_(ref) of the reference image 71 shown in FIG. 2. Asshown in FIGS. 5A to 5C, in cases where the driving voltage of theoscillation element 3 increases, a value of the liquid columnconstricted width w decreases. In cases where the driving voltage of theoscillation element 3 decreases, a value of the liquid columnconstricted width w increases. The controller 8 utilizes this relationto control the driving voltage of the oscillation element 3.

Similar to the above-mentioned first satellite upper interval d_(ref),the liquid column constricted width w sensitively changes with respectto the change in fluid droplet shape of the fluid stream S. Therefore,the fluid stream S can be maintained in a stable state, and also theposition of the break-off point BP can be stabilized by keepingadjusting the liquid column constricted width w so as to match with theliquid column constricted width w_(ref) of the reference image 71.

Note that, in controlling the driving voltage of the oscillation element3 by the controller 8, either the first satellite upper interval d orthe liquid column constricted width w may be an index. However, thefluid droplet shape of the fluid stream S can be further stabilized byusing both of them as indexes.

Alternatively, the driving voltage of the oscillation element 3 can becontrolled based only on the state of the fluid without using the stateof the satellite fluid droplet. FIG. 6 is a view showing a relationbetween the states of fluid, and the liquid column length L and a finalfluid droplet length m in the liquid column. FIGS. 7A to 7C are viewsshowing a relation between the driving voltage of the oscillationelement 3 and the final fluid droplet length m in the liquid column.

As shown in FIG. 6, a distance m (final fluid droplet length in theliquid column) from a position where the liquid column constricted widthw becomes minimum (narrowest part of the constricted region), to thebreak-off point has a close relation with the liquid column length L(the position of the break-off point BP). Therefore, the final fluiddroplet length m in the liquid column is an index which directly showsthe stability of the break-off point BP. Based on a value of the finalfluid droplet length m in the liquid column, the fluid droplet shape ofthe fluid stream S can be stabilized by controlling the driving voltageof the oscillation element 3.

More specifically, the driving voltage of the oscillation element 3 iscontrolled so that the value of the final fluid droplet length m in theliquid column in the image 72 during sorting becomes similar to a finalfluid droplet length m_(ref) in the liquid column of the reference image71 shown in FIG. 2. As shown in FIGS. 7A to 7C, in cases where thedriving voltage of the oscillation element 3 increases, the value of thefinal fluid droplet length m in the liquid column decreases. On theother hands, in cases where the driving voltage of the oscillationelement 3 decreases, the value of the final fluid droplet length m inthe liquid column increases. The controller 8 utilizes this relation tocontrol the driving voltage of the oscillation element 3.

In this manner, even in a case where no satellite fluid droplet isformed or a radius of an orifice is large, the fluid droplet shape ofthe fluid stream S can be stabilized by controlling the driving voltageof the oscillation element 3 with an index of the final fluid dropletlength m in the liquid column. Further, the final fluid droplet length min the liquid column is sensitive to the change in the fluid dropletshape of the fluid stream S. Therefore, fluid droplet formation duringsorting can be maintained as stable as the state similar to thereference image by keeping adjusting the final fluid droplet length m inthe liquid column so as to match the final fluid droplet length m_(ref)in the liquid column of the reference image 71.

Note that, for example, in cases where a plurality of the narrowestparts (positions where the liquid column constricted width w becomesminimum) exists in the constricted region of the fluid right beforebeing converted into fluid droplets, an arbitrary point, for example, acentral point of the constricted region or a point closest to thebreak-off point BP may be regarded as the narrowest part. Then, thefinal fluid droplet length m in the liquid column may be determined.Further, the final fluid droplet length m in the liquid column mayindependently be an index for controlling the driving voltage of theoscillation element 3. However, the driving voltage of the oscillationelement 3 can be controlled based on both the above-mentioned firstsatellite upper interval d and the liquid column constricted width w.

On the other hand, during sorting, there is a case where the fluiddroplet formation cannot be maintained stable and the break-off point BPdrastically drops because of the obstructions in the flow path andinterfusion of bubbles. However, such a situation can be detected basedon the liquid column length L. FIGS. 8A and 8B are schematic viewsshowing an anomaly detecting method based on the liquid column length L.In cases where an anomaly such as the obstructions in the flow path andinterfusion of bubbles occurs, the liquid column length L drasticallyincreases. Therefore, for example, a liquid column length L_(warn) fordetecting an anomaly is set in addition to a liquid column lengthL_(ref) as shown in FIG. 8A. When the liquid column length L exceedsthis value as shown in FIG. 8B, it is determined that “an anomaly hasoccurred”.

When the fluid droplet formation of the fluid stream S becomes unstable,sorting performance cannot be maintained. Therefore, in cases where ananomaly is detected, charging of the fluid droplets and application ofvoltage to the deflection plates are halted. Further, in such a case,sorting is stopped, and notification is given to a user. At the sametime, suction is carried out from the suction outlet 24 disposed in themicrochip 2. As a result, stability of the flow path (laminar flow) canbe achieved. In cases where the liquid column length L falls again belowthe liquid column length L_(warn) for detecting an anomaly, theabove-mentioned control process is carried out assuming that the flowpath (laminar flow) has been stabilized.

(Controlling Camera Position)

FIGS. 9A and 9B are views showing a changing status of the fluid streamdue to a change in environmental temperature. As shown in FIGS. 9A and9B, when a sheath liquid temperature changes due to the change in theenvironmental temperature during sorting, the fluid droplet intervals ofthe fluid stream S change because the flow speed changes due to a changein viscosity. Further, the position of the break-off point BP, that is,the liquid column length L changes as well. As a result, the number ofthe fluid droplets FD inside the liquid column in the image 72 changes.At the same time, there is a possibility that the break-off point BPcannot be stably detected or discriminated.

Under a condition in which the fluid droplet shape and pressure of thefluid stream S are stable, it can be considered that an influence on theliquid column length L is caused by the change in the fluid dropletintervals which is caused by the change in temperature. Accordingly, inthe microparticle sorting device 1 according to the embodiment, theposition of the camera 7 is moved with the controller 8 depending on thechange in the liquid column length L in the image. As a result, theposition of the break-off point BP in the image and the number of fluiddroplets FD inside the liquid column can be maintained at a constantlevel. Therefore, a drop delay time can be maintained at a constantvalue as well.

FIGS. 10A and 10B are views showing a method for moving the position ofthe camera 7 as the position of the break-off point changes. Forexample, in the method, the liquid column length L_(ref) can be obtainedfrom the reference image shown in FIG. 2. Further, as shown in FIGS. 10Aand 10B, when the liquid column length L in the image 72 during sortingexceeds a range of L_(ref)±m (m represents an arbitrary number of apixel), a position P of the camera 7 is controlled by the controller 8so as to negate the change in the liquid column length L.

In cases where the fluid droplet intervals broaden and the break-offpoint BP drops because of an increase in the flow speed due to anincrease in temperature, the value of the liquid column length Lincreases. Accordingly, the position of the camera 7 is lowered (P→P′).Further, in cases where the fluid droplet intervals have narrowed, theposition of the camera 7 rises (P′→P) in accordance with a decrease inthe liquid column length L.

In such a manner, when the position of the camera 7 is made to followthe change in the position of the break-off point BP, the value of theliquid column length L in the image can be maintained constant. As aresult, in a sorting image, the break-off point BP is stably maintainedat a predetermined position corresponding to the reference image.Therefore, the number of the fluid droplets FD inside the liquid columncan be maintained constant, and the drop delay time adjusted in advancecan be maintained for a long time.

As a method for constantly maintaining the position of the break-offpoint BP in the image other than a method for moving the camera 7itself, there is a method for changing an image cutting position. FIG.11 is a view showing other methods for making the position of thebreak-off point constant. For example, an image of fluid and fluiddroplets are taken with a wide-angle camera as shown in FIG. 11. Fromthe image, an image 73 including a break-off point BP is cut out so asto be used for control by the controller 8.

In such a case, when a position of the break-off point BP changes, theimage cutting position is also changed so as to control the change inthe value of the liquid column length L. As a result, it is possible tosimulate control of an imaging position as the break-off point BP moves.

The microparticle sorting device according to the present embodimentcontrols the driving voltage of the oscillation element and/or theposition of the imaging element based on the state of the fluid streamS. Therefore, it is possible to stabilize the fluid droplet shape for aprolonged period of time and to maintain the break-off point BP withhigh accuracy. The microparticle sorting device according to the presentembodiment performs control by using a parameter which sensitivelyreacts to the change in the fluid droplet shape. Therefore, the fluiddroplet shape can be controlled with high stability, fast-response, androbustness.

Further, in the microparticle sorting device according to the presentembodiment, stability of the flow path can be achieved by immediatedetection of the obstructions in the flow path and interfusion ofbubbles during sorting and emergency stop of sorting as well asautomatic suction inside the flow path. Further, in the microparticlesorting device according to the present embodiment, the position of thecamera 7 can follow the change in the sheath flow speed caused by thechange in the environmental temperature, and the change in the break-offpoint BP due to the change in the sheath flow speed. Accordingly, thenumber of the fluid droplets FD inside the liquid column to thebreak-off point BP can be maintained constant, and the drop delay timeadjusted in advance can be maintained for a long time, and also thesorting performance can be maintained.

As a result, according to the microparticle sorting device of thepresent embodiment, influences due to the change in the environmentaltemperature, a decrease of the sheath liquid/sample liquid, theobstructions and interfusion of bubbles, or the change in the fluiddroplet shape can be controlled. Therefore, it is possible to achievestable sorting with high accuracy over a prolonged period of time.

In the above-mentioned first embodiment, the example using the microchip2 has been described. However, the present disclosure is not restrictedthereto. Even in a case where a flow cell is used instead of themicrochip 2, a similar effect can be obtained. Further, the lightdetector of the present disclosure may be replaced with an electric ormagnetic detecting unit.

2. Second Embodiment

Next, a microparticle sorting device according to a second embodiment ofthe present disclosure will be described. FIG. 12 is a schematic view ofan overall configuration of the microparticle sorting device accordingto the second embodiment of the present disclosure. As shown in FIG. 12,the microparticle sorting device according to the present embodimentfurther includes a function of stabilizing pressure in addition to theconfiguration of the above-mentioned first embodiment.

The fluid droplet formation state of the fluid stream S discharged fromthe orifice 21 changes depending on the sheath pressure. FIGS. 13A and13B are views showing a relation between the sheath pressure and thestate of the fluid and the fluid droplets. As shown in FIGS. 13A and13B, when the sheath pressure is low, a position of the break-off pointBP rises. On the other hand, when the sheath pressure is high, theposition of the break-off point BP drops because the flow speedincreases.

Regarding sample flow, when sample pressure is low, an event rate (thenumber of detection per unit time) decreases. On the other hand, whenthe sample pressure is high, the event rate increases. In addition toair pressure controlled by compressors 13 and 18, pressure obtained byadding fluid pressure depending on a water depth is applied to an intakedisposed at the bottom of a sheath container 10 or a sample container15. In the microparticle sorting device according to the presentembodiment, to stabilize the pressure, a setting value of the airpressure is controlled depending on a change in the fluid pressure dueto a decrease in the water depth that occurs after sorting.

FIG. 14 is a schematic view showing the air pressure and water pressureinside the sheath container 10. FIG. 15 is a view showing a method formeasuring a water depth D_(sheath) of sheath liquid 231. FIG. 16 is aview showing a method for measuring a water depth D_(sample) of sampleliquid 221. FIGS. 17A and 17B are views showing a method for controllingthe sheath pressure. In the microparticle sorting device according tothe present embodiment, the water depth D_(sheath) of the sheath liquid231 and/or the water depth D_(sample) of the sample liquid 221 aremeasured.

As shown in FIGS. 14 and 15, the water depth D_(sheath) of the sheathliquid 231 can be calculated from the known mass and cross-sectionalarea of the sheath container 10 and from the density of the sheathliquid 231 by measuring the whole mass including the sheath container 10with a scale 11. On the other hand, as shown in FIG. 16, the water depthD_(sample) of the sample liquid 221 can be calculated by detecting abottom of the sample container 15 and a liquid surface of the sampleliquid 221 based on the image obtained by a camera 16, and by convertinga pixel distance between two points in the image to an actual distance.

Then, fluid pressure FP_(sheath) of the sheath liquid 231 and fluidpressure FP_(sample) of the sample liquid 221 are respectivelycalculated based on the water depth D_(sheath) of the sheath liquid 231and the water depth D_(sample) of the sample liquid 221. In such a case,the density of the sheath liquid 231 can be easily calculated based onthe known density. Further, the density of the sample liquid 221 isalmost similar to that of the sheath liquid 231 and can be calculatedfrom the density of the sheath liquid 231.

As shown in FIGS. 17A and 17B, a pressure controller 14 sets airpressure AP_(sheath) and gives an instruction to the compressor 13 tonegate a change in the fluid pressure FP_(sheath) due to a decrease inthe water depth D_(sheath), so that the sheath pressure P_(sheath)becomes constant. Herein, the sheath pressure P_(sheath) is equal to asum of the air pressure AP_(sheath) and the fluid pressure FP_(sheath)(P_(sheath)=AP_(sheath)+FP_(sheath)). Therefore, the sheath pressureP_(sheath) can be stabilized for a prolonged period of time without anychange by periodically calculating the fluid pressure FP_(sheath) andsetting the air pressure AP_(sheath) in accordance with the calculatedfluid pressure FP_(sheath).

Further, the pressure controller 14 can control the sample pressureP_(sample) instead of controlling the above-mentioned sheath pressureP_(sheath), or together with controlling the sheath pressure P_(sheath).Even in such a case, similar to the above-mentioned sheath pressureP_(sheath), the pressure controller sets the air pressure AP_(sample)depending on the fluid pressure FP_(sample) and gives an instruction tothe compressor 18. As a result, the sample pressure P_(sample) can bestabilized for a prolonged period of time.

In the microparticle sorting device according to the present embodiment,the air pressure is adjusted so that a sum of the air pressure and thefluid pressure in the sheath liquid becomes constant. Therefore, thechange in the fluid pressure due to the decrease in the water depth ofthe sheath liquid is controlled, and it is possible to achieve constantand stable sheath flow for a prolonged period of time. Further, in themicroparticle sorting device according to the present embodiment, thechange in the air pressure and in the fluid pressure of the sampleliquid is controlled together with controlling the sheath pressure, orinstead of controlling the sheath pressure. Therefore, it is possible toachieve sorting which maintains a constant event rate for a prolongedperiod of time.

Note that configurations and effects other than those describedhereinbefore of the microparticle sorting device according to thepresent embodiment are similar to those described in the above-mentionedfirst embodiment.

Further, the present disclosure may have configurations hereinafterdescribed.

(1)

A microparticle sorting device including:

an imaging element configured to obtain an image of fluid and fluiddroplets at a position where the fluid discharged from an orifice whichgenerates a fluid stream is converted into the fluid droplets; and

a controller configured to control driving voltage of an oscillationelement which gives oscillation to the orifice and/or control a positionof the imaging element, based on a state of the fluid in the imageand/or a state of a satellite fluid droplet which does not includemicroparticles and exists between the position, where the fluid isconverted into the fluid droplets, and a fluid droplet, among fluiddroplets including the microparticles, which is closest to the positionwhere the fluid is converted into the fluid droplets.

(2)

The microparticle sorting device according to (1), wherein thecontroller controls the driving voltage such that a distance from theposition, where the fluid is converted into the fluid droplets, to thesatellite fluid droplet and/or a state of a constricted region of thefluid right before being converted into the fluid droplets becomeconstant.

(3)

The microparticle sorting device according to (2), wherein thecontroller controls the driving voltage such that a width of theconstricted region becomes constant.

(4)

The microparticle sorting device according to (1), wherein thecontroller controls the driving voltage such that a distance from theposition, where the fluid is converted into the fluid droplets, to anarrowest part of the constricted region of the fluid right before beingconverted into the fluid droplets becomes constant.

(5)

The microparticle sorting device according to any one of (1) to (4),wherein the controller controls the position of the imaging element suchthat the position, in the image, where the fluid is converted into thefluid droplets becomes constant.

(6)

The microparticle sorting device according to (5), wherein thecontroller calculates a distance from an upper end of the image to theposition where the fluid is converted into the fluid droplets, andcontrols the position of the imaging element such that the distancebecomes constant.

(7)

The microparticle sorting device according to any one of (1) to (6),including:

a sheath liquid storage tank configured to store sheath liquid includedin the fluid stream;

a first water depth detector configured to detect a water depth of thesheath liquid stored in the sheath liquid storage tank;

a first pressure detector configured to detect air pressure inside thesheath liquid storage tank; and

a first pressure controller configured to control the air pressureinside the sheath liquid storage tank such that a sum of the fluidpressure which is calculated from the water depth detected by the firstwater depth detector and the air pressure detected by the first pressuredetector becomes constant.

(8)

The microparticle sorting device according to any one of (1) to (7),including:

a sample liquid storage tank configured to store sample liquid includingmicroparticles and included in the fluid stream;

a second water depth detector configured to detect a water depth of thesample liquid stored in the sample liquid storage tank;

a second pressure detector configured to detect air pressure inside thesample liquid storage tank; and

a second pressure controller configured to control the air pressureinside the sample liquid storage tank such that a sum of the fluidpressure which is calculated from the water depth detected by the secondwater depth detector and the air pressure detected by the secondpressure detector becomes constant.

(9)

A method for sorting microparticles, including:

controlling driving voltage of an oscillation element which givesoscillation to an orifice and/or a position of an imaging element thatobtains an image based on a state of fluid in the image taken at aposition where the fluid discharged from the orifice which generates afluid stream is converted into fluid droplets and/or a state of asatellite fluid droplet which does not include microparticles and existsbetween the position, where the fluid is converted into the fluiddroplets, and a fluid droplet, among the fluid droplets including themicroparticles, which is closest to the position where the fluid isconverted into the fluid droplets.

(10)

The method for sorting microparticles according to (9), wherein thedriving voltage is controlled such that a distance from the position,where the fluid is converted into the fluid droplets, to the satellitefluid droplet and/or a state of a constricted region of the fluid rightbefore being converted into the fluid droplets become constant.

(11)

The method for sorting microparticles according to (10), wherein thedriving voltage is controlled such that a width of the constrictedregion becomes constant.

(12)

The method for sorting microparticles according to (9), wherein thedriving voltage is controlled such that a distance from the position,where the fluid is converted into the fluid droplets, to a narrowestpart of the constricted region of the fluid right before being convertedinto the fluid droplets becomes constant.

(13)

The method for sorting microparticles according to any one of (9) to(12), wherein the position of the imaging element is controlled suchthat the position, in the image, where the fluid is converted into thefluid droplets becomes constant.

(14)

The method for sorting microparticles according to (13), wherein adistance from an upper end of the image to the position where the fluidis converted into the fluid droplets is calculated, and the position ofthe imaging element is controlled such that the distance becomesconstant.

(15)

A program which causes a controller of a microparticle sorting device toexecute a function of controlling driving voltage of an oscillationelement which gives oscillation to an orifice and/or a position of animaging element which obtains an image, based on a state of fluid in theimage taken at a position where the fluid discharged from the orificewhich generates a fluid stream is converted into fluid droplets and/or astate of a satellite fluid droplet which does not include microparticlesand exists between the position, where the fluid is converted into thefluid droplets, and a fluid droplet, among the fluid droplets includingthe microparticles, which is closest to the position where the fluid isconverted into the fluid droplets.

REFERENCE SIGNS LIST

-   1 microparticle sorting device-   2 microchip-   3 oscillation element-   4 electrode for charging-   5 a, 5 b deflection plate-   6 a to 6 c recovery container-   7 imaging element (camera)-   8 controller-   10 sheath container-   11 scale-   12, 17 air pressure sensor-   13, 18 compressor-   14 pressure controller-   15 sample container-   16 camera-   21 orifice-   22 sample inlet-   23 sheath inlet-   24 suction outlet-   31 voltage supplier-   70 position adjustment mechanism-   71 to 73 image-   221 sample liquid-   231 sheath liquid-   BP break-off point-   D fluid droplet-   S fluid stream-   SD satellite fluid droplet-   FD fluid droplet inside liquid column-   L liquid column length-   m final fluid droplet length in liquid column-   w liquid column constricted width

1. A microparticle sorting device, comprising: an imaging elementconfigured to obtain an image of at least one part of a fluid streamincluding fluid and fluid droplets; and a controller configured tocontrol a driving voltage of an oscillation element which givesoscillation to an orifice and to control a position of the imagingelement, based on a distance from a break-off point where the fluid isconverted into the fluid droplets to a first satellite fluid dropletwhich does not include microparticles.